Seymour P. Halbert

Immunochemical analyses of lens protein separations.

Abstract Continuous flow electrophoresis, column chromatography, and isoelectric and ethanol precipitation were used for the separation of soluble lens proteins. The fractions obtained were analyzed by immunoelectrophoresis or immune double-diffusion precipitation. Almost all the fractions proved to be mixtures of greater or lesser complexity. A total of at least ten components were found. It has been shown that components with the same immunological specificity occur in a heterogeneity of forms different in solubility and electrophoretic and Chromatographic properties. These data indicate that interpretation of separation patterns based only on the occurrence of protein peaks may be misleading and point to the necessity of immunochemical analysis as an accessory procedure. The possible roles of protein interactions, aging, and genetic variations as causes of difficulty in the separation of individual lens proteins are briefly discussed.

IMMUNOLOGIC AND BIOCHEMICAL COMPARISON OF URINARY GLYCOPROTEINS IN PATIENTS WITH CYSTIC FIBROSIS OF THE PANCREAS AND NORMAL CONTROLS.

Urinary paper-filtered, nonultrafiltrable macromolecules from patients with cystic fibrosis and normal children were compared in an effort to find biochemical or immunologic differences. Fractionation by continuous flow electrophoresis and DEAE-cellulose chromatography followed by analysis for carbohydrate moieties, protein content, proteolytic activity, and ability to inhibit viral hemagglutination, revealed minor differences. With the use of rabbit antisera with immunodiffusion in agar, the urinary antigens were found immunologically identical in normal and pathologic urine concentrates. Of the serum proteins detectable in urine, transferrin was absent or decreased in all pathologic samples tested, but was readily found in 10 of 12 normal urinary concentrates.

Comparison of rabbit antilens antibodies indused by iso- and heteroimmunization.

The immunological organ specificity of the lens tissue, or its wide extent of taxonomic cross reactions, has been known and explored for more than half a century.’ ’ I t also has been known that antivertebrate lens sera obtained by heterologous immunization contain antibodies reactive with pooled lenses from the animal species used for immunization,’.’ and tha t similar antibodies are relatively easily produced by homologous immunization.’ I t will be shown that in both instances the antibodies formed can be properly defined as autoantibodies, i.e. can react with the lens antigens of the individual animal which has synthesized the antibodies. A comparison of the lens autoantibodies produced by heteroand isoimmunization is described below. Examples of the reaction between antivertebrate lens sera and pooled lenses from the species used for immunization are presented in FIGURE 1. The photographs show the immunoelectrophoretic patterns obtained by rabbit antichicken and rabbit antibovine lens sera with rabbit lens, and by duck antirabbit lens serum with duck lens. The reactions of the same immune sera with bovine and chicken lenses are included for comparison with another species from the same class. In all the immunoelectrophoretic experiments reported here, microscope slides (1 x 3 inches) were covered with 2.5 ml. of 2 per cent agar in veronal buffer of pH 8.2 and p 0.05.’ Lenses were homogenized and the insoluble part discarded after centrifugation. Two per cent lens protein solution in the above veronal buffer were used. A direct current of 6 V/cm. and 2.5 mA. per slide was applied for 90-120 min. Preparation of antigens and antisera electrophoresis and development were performed in the cold at 4°C. Photographs were taken after 72-96 hours exposure to the immune serum. The anode is placed in all photographs on the left side.’ The immunoelectrophoretic reactions of rabbit lens with rabbit antibovine or rabbit antichicken lens sera showed similar numbers and patterns of precipitin arcs to those given by bovine lens (FIGURE la and b) . Other rabbit antivertebrate lens sera revealed analogous results (e.g. FIGURES 2 and 4). Duck antirabbit lens serum gave immunoelectrophoretic patterns with rabbit lens also similar to those given by lenses from other mammals (e.g. FIGURE l c ) . The minimum number of antibodies to different rabbit lens proteins produced by the rabbit was, however, significantly greater when bovine rather than chicken lens was used for immunization (FIGURE la , b).

Naturally occurring human antibodies to streptococcal enzymes.

It has long been known that streptococci can secrete an array of enzymes, e.g., hyaluronidase, desoxyribonuclease, proteinase, ribonuclease, lipoproteinase, and diphosphopyridine nu~leotidase.l~-~7 However, the discovery of these streptococcal enzymes and other antigens has been on a chance basis until recently. The development of immunodiffusion technics has opened the way for a rational analysis of the total number of antigens released by the organisms in vivo, as reflected by the number of antibody responses detected. With such an approach, the sera of patients with streptococcal disease have been found to contain numerous antibodies to extracellular streptococcal products, but to contain few if any to cellular components from the same strain.l~2JsJ9 Even patients without recent history of streptococcal disease were shown to contain variable, but usually small numbers of precipitating antibodies to the same extracellular antigens. Tests then made with pooled and concentrated normal human gamma globulinf solutions revealed that it was extraordinarily rich in such anti-streptococcal antibodies. An immunoelectrophoretic test of crude culture supernate concentrates of group A streptococcal growth against human gamma globulin revealed at least 20 extracellular products which must have been secreted in vivo.6 An example of these findings is shown in FIGURE 1, a1 and a2, in which two different culture concentrates from the C203S group A streptococcal strain and a group C streptococcal concentrate were separated electrophoretically, and then exposed to the antibodies in pooled human gamma globulin. In FIGURE l b is shown a diagrammatic representation of the results obtained with the crude streptococcal concentrate which was used as a reference standard system. The reproducibility of the general patterns observed was quite great, and high concentrations of the culture concentrates appeared essential.